Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase
Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase
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DOI：
10.1038/nature01083
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发表时间：
2002-10-10
期刊：
影响因子：
64.8
通讯作者：
Chu, S
中科院分区：
文献类型：
--
作者：
Ha, T;Rasnik, I;Chu, S
解旋酶是将由ATP结合和水解诱导的构象变化与双链体核酸的解旋偶联的马达蛋白（1-3），并且涉及多种人类疾病。一些作为六聚体环发挥功能（4），但非六聚体解旋酶的功能形式一直存在争议（5-10）。在这里，我们使用的表面固定化方案和单分子荧光分析的组合，这不干扰生物活性的探测DNA解旋的大肠杆菌Rep解旋酶。我们的研究表明，Rep单体利用ATP水解向单链和双链DNA之间的连接处移动，但随后显示出不会导致DNA解旋的构象波动。DNA解旋只有在通过额外的蛋白质结合形成功能性解旋酶时才开始。在解旋过程中功能性复合物的部分解离导致中断（“停滞”），其导致复合物完全解离后的双链体重绕，或导致功能性解旋酶重新形成后的解旋重新开始。这些结果表明，在体外观察到的Rep的低解旋持续合成能力是由于功能复合物的相对不稳定性。我们希望这些技术将是有用的其他解旋酶和蛋白质-DNA相互作用的动态研究。
Helicases are motor proteins that couple conformational changes induced by ATP binding and hydrolysis with unwinding of duplex nucleic acid(1-3), and are involved in several human diseases. Some function as hexameric rings(4), but the functional form of non-hexameric helicases has been debated(5-10). Here we use a combination of a surface immobilization scheme and single-molecule fluorescence assays-which do not interfere with biological activity-to probe DNA unwinding by the Escherichia coli Rep helicase. Our studies indicate that a Rep monomer uses ATP hydrolysis to move toward the junction between single-stranded and double-stranded DNA but then displays conformational fluctuations that do not lead to DNA unwinding. DNA unwinding initiates only if a functional helicase is formed via additional protein binding. Partial dissociation of the functional complex during unwinding results in interruptions ('stalls') that lead either to duplex rewinding upon complete dissociation of the complex, or to re-initiation of unwinding upon re-formation of the functional helicase. These results suggest that the low unwinding processivity observed in vitro for Rep is due to the relative instability of the functional complex. We expect that these techniques will be useful for dynamic studies of other helicases and protein-DNA interactions.